For a number of medical reasons, it can be desirable to monitor the level of sugars, in particular glucose, in the bloodstream. This is particularly important in the diagnosis and treatment of diabetics, and also for patients in intensive care, where it has been found that changes in blood glucose levels can provide vital information about potential health complications.
Diabetes is a growing medical problem, currently thought to affect about 5% of the global population. Although control is possible through lifestyle management and/or insulin injections, serious issues remain. A low blood glucose concentration, caused by excess insulin, can be fatal, whilst high glucose levels can lead to long term complications such as heart disease, blindness, kidney damage, stroke and nerve damage.
Close control of blood glucose levels is therefore desirable for both diabetics and intensive care patients. Ideally, such control would involve the accurate and continuous (or at least timely) measurement of blood glucose concentrations. However, whilst periodic analyses of withdrawn blood samples are routine, continuous monitoring remains an unsolved problem. Some systems have reached the market-place, but their reliance on enzyme-based detection technology imposes limitations: in particular, they only measure glucose concentrations in interstitial fluid, just beneath the skin, and these lag behind the more important blood glucose concentrations.
Were such problems to be solved in a practical manner, this could assist in the design of an “artificial pancreas”, which could continuously supply insulin to a patient's bloodstream in response to changes in blood glucose levels, in order to maintain those levels within a desired, safe range. Such systems could prove life-changing for diabetics and their careers. The ability to monitor blood glucose levels continuously, in vivo, could also significantly improve the care of patients in intensive care, and potentially of other at-risk individuals.
Historically, the detection of saccharides in an aqueous environment such as blood has presented challenges. Saccharides are hydrophilic species, bearing hydromimetic hydroxyl groups, which makes them difficult to extract from water. For a chemical detection system, distinguishing between target molecule and solvent is a significant problem. Achieving selectivity for a specific target molecule is also non-trivial: the generic carbohydrate structure allows great scope for variation, but differences between individual saccharides are often subtle (for example, the configuration of a single asymmetric centre).
As referred to above, it is known to assay blood for glucose levels using enzymes, for example glucose oxidase, which bind selectively to glucose molecules and thereby generate a detectable electrochemical signal. Such techniques usually have to be carried out on isolated fresh blood samples withdrawn from a patient's body, rather than in vivo, and they also result in destruction of the glucose they detect; they do not therefore lend themselves to continuous blood glucose monitoring. Typically a power source is required for detection of the enzyme-glucose interaction, and moreover enzymes tend to have poor stability. Receptor-based approaches are therefore likely to prove more suitable for glucose monitoring, but none have yet been approved for general use.
Thus far, most work on receptor-based glucose sensing has employed boronic acids, which bind to carbohydrates through covalent B—O bonds. These receptors may also incorporate chromophore labelling moieties, to allow their detection for instance by fluorescence spectroscopy. It is already known to introduce such labelled boronic acid-based receptors into the bloodstream in the form of a coating on a probe such as a fibre optic cable, for use in the continuous monitoring of blood glucose levels. However, boronic acid-based receptors tend to have a relatively low selectivity for glucose: they can also bind to other carbohydrates, and to diols and lactates, which may be present in the bloodstream. They can also be sensitive to oxygen, which again can compromise their efficacy as glucose receptors in the bloodstream.
Lectins are naturally-occurring proteins which are capable of binding to saccharides, and as such they too have been used to assess blood glucose levels in particular in medical diagnostic techniques. Examples of lectins used in this way include Concanavalin A, Lens culinaris agglutinin and Pisum aativum agglutinin. Even these, however, tend to show low affinities for the target saccharides, and often quite modest selectivities. Research has therefore turned to the creation of synthetic analogues. Synthetic lectins are organic molecules which are capable of biomimetic saccharide recognition, ie binding to saccharides in aqueous systems using the non-covalent interactions employed by natural lectins.
Perhaps unsurprisingly, due to the hydrophilicity and stereochemical complexity of carbohydrates, the design of synthetic lectins has proved to be less than straightforward. Although progress has been made, binding affinities have been mostly low and good selectivities rare. Moreover, success usually comes at the cost of structural complexity.
The octalactam 2 shown in FIG. 1A, reported previously by Barwell et al [Angew Chem, Int Ed, 48; 7673-7676 (2009)], is an example of a synthetic lectin analogue proposed for use in the detection of carbohydrates. This tricyclic system is able to surround a β-D-glucose molecule 1, providing polar and apolar surfaces which complement the all-equatorial substitution pattern of the carbohydrate. Complex formation is thought to be driven by hydrophobic CH-π interactions between saccharide CH and biphenyl surfaces, and by polar interactions between saccharide OH groups and isophthalamide groups in compound 2. The propoxy groups (—OPr) appear to be required for optimal glucose selectivity.
The compound 2 shows excellent selectivity for glucose; for example, ratios of binding constants are 20:1 for glucose vs galactose, and 60:1 for glucose vs mannose. The affinity of the lactam for glucose is 60 M−1, which may seem low, but is actually state-of-the-art for a synthetic system operating in water through non-covalent interactions: the well-studied lectin Concanavalin A is just one order of magnitude stronger. Furthermore, the affinity is not too low to be useful, in particular in the detection of glucose in blood: as blood glucose levels are relatively high (˜2-13 mM), binding affinities need to be moderate to avoid receptor saturation.
Synthetic lectins such as 2 are therefore promising, but their elaborate structures can impose a barrier to further development. The oligoamide 2 is designed to enclose its carbohydrate target, providing complementary surfaces as shown in FIG. 1A. Though apparently the key to success, this results in a complex cage architecture, requiring a 20-step synthesis with an overall yield of just ˜0.1%. Preparing substantial quantities can be difficult, and further modification (for example to link the receptor to a substrate surface) represents a major undertaking.
The synthetic lectin 2 possesses a further potential disadvantage for practical glucose sensing. Receptor-based sensing requires a signalling system, to allow measurement of the level of occupancy by the target molecule. Receptor 2 presents no clear opportunities in this respect.
We have now been able to create a novel class of glucose receptor compounds, which can overcome or at least mitigate the above described problems. Embodiments of the invention can allow the efficient and selective detection of blood glucose levels, in vivo, using optical signals. They can thus be of use in continuous blood glucose monitoring. Moreover, these new compounds can be significantly less complex than the previously reported synthetic lectins, making them more simple and inexpensive not only to prepare, but also to tailor for use in a specific environment or physical form, or for a specific purpose.